Institution: | 1. Department of Applied Chemistry and Biological Engineering, Ajou University, Suwon, 16499, Republic of Korea;2. Department of Molecular Science and Technology, Ajou University, Suwon, 16499, Republic of Korea;1. Department of Histology and Embryology, School of Basic Medical Sciences, Zhengzhou University, Zhengzhou 450001, Henan, China;2. Department of Biochemistry and Molecular Biology, Xinxiang Medical University, Xinxiang 453003, Henan, China;3. Henan Engineering Research Center for Biopharmaceutical Innovation, Xinxiang 453003, Henan, China;1. Department of Biological Engineering, Inha University, 100 Inha-ro, Michuhol-gu, Incheon 22212, Republic of Korea;2. Biotherapeutics and Glycomics Laboratory, College of Pharmacy, Chung-Ang University, 84 Heukseok-ro, Dongjak-gu, Seoul 06974, Republic of Korea;3. Department of Global Innovative Drug, The Graduate School of Chung-Ang University, 84 Heukseok-ro, Dongjak-gu, Seoul 06974, Republic of Korea;4. Department of Life Sciences, Department of Health Sciences and Technology, GAIHST, Gachon University, Seongnam-daero, Sujeong-gu, Seongnam 13120, Republic of Korea;1. Department of Biological Sciences, KAIST, Daejeon, Republic of Korea;2. The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Lyngby, Denmark;1. The Novo Nordisk Foundation, Center for Biosustainability, Technical University of Denmark, Kongens, Lyngby, Denmark;2. Department of Biotechnology and Biomedicine, Technical University of Denmark, Kongens, Lyngby, Denmark |
Abstract: | Sialic acid, a terminal monosaccharide present in N-glycans, plays an important role in determining both the in vivo half-life and the therapeutic efficacy of recombinant glycoproteins. Low sialylation levels of recombinant human erythropoietin (rhEPO) in recombinant Chinese hamster ovary (rCHO) cell cultures are considered a major obstacle to the production of rhEPO in fed-batch mode. This is mainly due to the accumulation of extracellular sialidases released from the cells. To overcome this hurdle, three sialidase genes (Neu1, 2, and 3) were initially knocked-out using the CRISPR/Cas9-mediated large deletion method in the rhEPO-producing rCHO cell line. Unlike wild type cells, sialidase knockout (KO) clones maintained the sialic acid content and proportion of tetra-sialylated rhEPO throughout fed-batch cultures without exhibiting a detrimental effect with respect to cell growth and rhEPO production. Additional KO of two pro-apoptotic genes, BAK and BAX, in sialidase KO clones (5X KO clones) further improved rhEPO production without any detrimental effect on sialylation. On day 10 in fed-batch cultures, the 5X KO clones had 1.4-times higher rhEPO concentration and 3.0-times higher sialic acid content than wild type cells. Furthermore, the proportion of tetra-sialylated rhEPO on day 10 in fed-batch cultures was 42.2–44.3% for 5X KO clones while it was only 2.2% for wild type cells. Taken together, KO of sialidase and pro-apoptotic genes in rCHO cells is a useful tool for producing heavily sialylated glycoproteins such as rhEPO in fed-batch mode. |